Fibrosis is the final common pathway to organ failure in many chronic diseases, leading to over $142 billion in annual medical costs in the United States. Intestinal fibrosis drives the need for surgery in Crohn's disease, which is required in two-thirds of all patients. Despite this immense impact on U.S. healthcare and patients with Crohn's disease, we have no medical therapies for intestinal fibrosis. Our preliminary data show that small molecules targeting the Bcl-2 signaling pathway can inhibit the activation of intestinal fibroblasts. The objective of this proposal is to validate Bcl-2 signaling as an important pathway for intestinal fibrosis. The long-term objective of this research program is to develop inhibitors of Bcl-2 that will lead to effective medical therapies for intestinal fibrosis in Crohn's disease. Our preliminary data demonstrate that candidate Bcl-2 inhibitors inhibit human intestinal myofibroblast activation in vitro and in human intestinal organoids. However, these compounds have limited potency, and potential toxicity from systemic exposure in mice. An existing Bcl-2 inhibitor, navitoclax, has been proven safe in humans and is now in phase 2 clinical trials for cancer. We propose to improve upon the current compounds to enhance compound potency and gut specificity to reduce systemic toxicity. In Aim 1, we will iteratively develop and test novel variants of these compounds for in vitro target specificity and anti-fibrotic activity. Potency will be shown in two independent models of myofibroblast activation, using TGF? (cytokine activation) and matrix stiffness (mechanical activation). In Aim 2, we will optimize the pharmacokinetic properties of the most promising Bcl-2 inhibitors from Aim 1 by evaluating oral absorption into the gut and compound instability in plasma and liver microsomes. In Aim 3, we will determine the maximum tolerated dose of promising candidate compounds, and perform PK and PD testing in mice. Promising gut-selective compounds will progress to stepwise testing of the biologic efficacy of these compounds in two rodent models, the Salmonella typhimurium mouse model of intestinal fibrosis, and the rat trinitrobenzene sulfonic acid (TNBS) model of intestinal fibrosis. The proposed studies are innovative in that they will generate gut-selective compounds that inhibit a novel antifibrotic pathway, and rigorously test the best candidates in two rodent models. This research proposal is significant in that it has the potential to impact patient care by validating this pathway in organ fibrosis, a significant unmet clinical need in Crohn's disease and in most forms of chronic organ failure, including kidney failure and liver cirrhosis.